Method for the determination of serum acid phosphatase and diagnostic preparation therefor



United States Patent METHOD FOR THE DETERMINATION OF SERUM ACIDPHOSPHATASE AND DIAGNOSTIC PREP- ARATION THEREFOR Arthur L. Babson,Morris Plains, NJ., assignor to Warher-Lambert Pharmaceutical Company,Morris Plains, NJ., a corporation of Delaware No Drawing. Filed Nov. 13,1958, Ser. No. 773,571

13 Claims. (Cl. 195103.5)

This invention relates to a new and improved process for thedetermination of serum acid phosphatase and relates more particularly toa stable composition for use in a simplified semi-quantitativedetermination of acid phosphatase in serum.

Acid phosphatase is an enzyme which has the ability to hydrolyzephosphate esters in an acid medium (pH 5). This enzyme is present in theprostate gland of humans and, in cases of cancer of the prostate, theenzyme is released with the result that the serum acid phosphatase levelincreases several fold above normal values. For this reason a reliabletest for serum acid phosphatase activity levels is of great value as adiagnostic aid in detecting cancer of the prostate. A method for thedetermination of acid phosphatase in serum was developed by Shinowara eta1. and reported in the Journal of Biological Chemistry, vol. 142, page921 (1942). This method employs a bufiered glycerophosphate solution anddepends on the release of phosphate ion in the serum to be tested as aresult of activity of the acid phosphatase enzyme. The releasedphosphate ion is determined bya colorimetric procedure which relies uponthe conversion of the phosphate ion to a colored phosphomolbdatecomplex.

This method has several disadvantages which render it generallyunacceptable as a rapid diagnostic test unless the test is carried outby trained personnel under carefully controlled conditions. The volumeof the serum and of all test reagents must be measured accurately and atleast two (2) ml. of serum is required. In addition, an incubationperiod of one hour is required. Also, the effectiveness of this testdepends upon a measurement of the released phosphate ion which is formedby the activity of the acid phosphatase enzyme on the butteredglycerophosphate solution. However, serum normally contains anappreciable quantity of phosphate ion and the phosphate ion releasedfrom the glycero phosphate by the action of the enzyme will, in mostcases, be only a small proportion of the phosphate ion normallyoccurring in the serum. For this reason, it is necessary to carry out ablank determination on the serum without the addition of bufferedglycerophosphate in order to determine the phosphate ion normallypresent in the serum and the acid phosphatase enzyme activity is thendetermined by observing the dilference in color between the blankdetermination and the determination made with the addition of butteredglycerophosphate. Since 'any colorimetric determination is subject tocertain inaccuracies, it is apparent that this method must be carriedout with great care and precision to obtain any worthwhile anddependable results.

It is an object of this invention to provide a method of determiningserum acid phosphatase which is capable of yielding reliable resultswithout any necessity for highly skilled laboratory technicians.

It is a further object of the invention to provide a method fordetermining serum acid phosphatase where only a small volume of serum isrequired and where the need for precise and time-consuming measurementsis eliminated.

It is a still further object of the invention to provide a substrate foruse in the determination of serum acid phosphatase which is specific toacid phosphatase released by the prostate gland. Other objects and theadvantages of the invention will appear hereinafter.

In accordance with the present invention, serum acid phosphatase isdetermined in a small volume of serum by a process in which the serum isincubated with a substrate comprising a solid preparation consisting ofa metal salt of a phenolic phosphate ester and a buffer, and afterincubation, an azonium salt of an aromatic amine containing from 1 to2-NH groups per molecule is added and the color which then develops iscompared with a standard calibrated color chart, the depth of colorobserved being a measure of the prostatic acid phosphatase activity inthe serum being tested.

In accordance with a preferred embodiment of this invention, I employ asubstrate comprising a buffer and a salt of ot-naphthyl phosphate. Thissubstrate has been found to be specific to serum acid phosphatasereleased by the prostate gland and when test serum is incubated withthis substrate and the azonium salt added, prostatic acid phosphatase isreadily determined.

In the method of this invention, the acid phosphatase enzyme present inthe serum hydrolyzes the substrate Which liberates phosphate ion andfree phenolic compound, which then couples with the azonium salt to forman azo dye. Since the procedure does not depend on the colorimetricdetermination of liberated phosphate ion, it is not necessary to run ablank on each sample of serum tested to determine the phosphate ionnormally present. Serum does not contain any free phenolic compound, sothat any color developed during the test must be due solely to activityof acid phosphatase. The simplicity of the method of the inventioneliminates the need for expensive equipment and highly trained technicalpersonnel to perform the test. The simplicity and rapidity of theprocedure renders the test valuable for the screening of serum acidphosphatase levels in large population groups and furnishes a convenientand inexpensive means for early diagnosis of cancer of the prostate.

The acid phosphatase enzyme which is present in the prostate gland isprostatic acid phosphatase which diifers from the acid phosphatasenormally present in red blood cells, which is erythrocytic acidphosphatase. In cases where cancer of the prostate exists, prostaticacid phosphatase is released into the serum which will not normallycontain any erythrocytic acid phosphatase. There is, however, always apossibility that as a result of hemolysis of the blood sample taken,some erythrocytic acid phosphatase may be released into the serum andthe presence of this enzyme will give a false prostatic acid phosphatasereading. It would be desirable to have a test which is specific toprostatic acid phosphatase to eliminate the possibility of misleadingresults should the serum tested contain any erythrocytic acidphosphatase.

It has been found that the metal salts of u-naphthyl phosphate, forexample, sodium a-naphthyl phospate, are specific in their property ofbeing hydrolyzed far more rapidly by prostatic acid phosphatase than byerythrocytic acid phosphatase. It is for this reason that the salts ofa-naphthyl phosphate are preferred ingredients in the substrate sinceany acid phosphatase activity determined is that of prostatic acidphosphatase. The use of this reagent eliminates the possibility thatcontamination of the sample with erythrocytic acid phosphatase willcause misleading results.

The preferred substrate also contains a buffer soluble in serum in anamount sufficient to maintain in the serum a pH of about 4.5 to 5.5which is the optimum pH for acid phosphatase enzyme activity. In orderto obtain reproducible results it is necessary that this pH bemaintained in the serum throughout the entire test procedure. Citratebutters based on citric acid are particularly cffective, such as amixture of citric acid and trisodium citrate, ,a mixture of disodiumcitrate and trisodium citrate, or disodium citrate alone. Salts ofoxalic acid or malic acid can also be used as buffers. Salts of tartaricand phosphoric acids interfere with the acid phosphatase inducedhydrolysis and should be avoided.

The quantities of the a-naphthyl phosphate salt and the buffer in thesubstrate can be varied within wide limits provided that there is anexcess of the salt present. In addition there should be suflicientbuffer so that the pH can be maintained in the critical range of about4.5 to 5.5 throughout the incubation period. The presence in thesubstrate of about 0.4 to about 1 mg. of a-naphthyl phosphate salt andabout to about 25 mg. buffer is a particularly effective range and willnormally provide an excess of salt and effective buffering with thenormal volumes of serum tested.

The substrate can be in the form of a liquid, with the v a-naphthylphosphate salt and buffer in solution in a solvent such as water. It ispreferred that the substrate be formulated as a solid mixtureofa-naphthyl phosphate salt and buffer which can be added to a smallvolume of serum in carrying out the test without the necessity fortime-consuming measurements of liquid volumes. A substrate in the formof a tablet is a particularly convenient form. In preparing the tablet,the active ingredients comprising the a-naphthyl phosphate salt and thebufier are mixed with conventional tableting excipients includingfillers such as lactose, sucrose and the like together with lubricantssuch as leucine, Carbowax-6000 and the like. Carbowax-6000 is a solidpolyethylene glycol having a molecular weight of about 6000 and amelting point of about 60 C. produced by Carbide and Carbon Chemicals,South Charleston, West Virginia. Although salts of a-naphthyl phosphateare preferred, other phenolic phosphate ester salts can be used to forma solid substrate for use in the invention. Thus, the alkail andalkaline earth metal salts of a phosphoric acid ester of ahydroxy-substituted aromatic compound selected from the group consistingof benzene and naphthalene can be used. Suitable salts include the saltsof sodium, potassium, lithium, barium calcium and magnesium, forexample, but the alkali metal salts are preferred because of their moreready solubility in serum. The phosphoric acid esters of aromatichydroxy compounds such as anaphthol, fl-naphthol, ortho-, metaandp-cresol, phenol, catechol, resorcinol, hydroquinone and the like havebeen found to be quite useful.

The formulation of a phenolic phosphate ester salt such as describedabove with a bulfer into a solid substrate is particularly effective incarrying out the test in accordance with this invention since nomeasurements of liquid volumes need be made. Where a salt of aphosphoric acid ester of a hydroxy substituted aromatic compound otherthan u-naphthol is used to form a solid substrate, care must be takenthat no hemolysis of the blood sample to be tested takes place, sincethe phenolic phosphate ester salts other than a-naphthyl phosphate saltsare hydrolyzed by erythrocytic acid phosphatase.

In carrying out the actual diagnostic test, a mixture of serum andsubstrate is incubated for a predetermined or standard time periodcausing the phosphate ester to be split by the activity of the acidphosphatase enzyme thus releasing the hydroxy substituted aromaticcompound forming the ester. In the case of the preferred a-naphthylphosphate salt, of course, a-naphthol is released. The incubation periodemployed will vary with the temperature. At a temperature of 65 F. theincubation period should be about 25 minutes; at 72 F. about minutes, at86 F. about 15 minutes and at 98 F. about 12 minutes.

The incubation need not be carried out in complex temperature controlledbaths, but can be carried out at normal room temperature with the timebeing adjusted depending on the temperature of the room.

At the conclusion of the incubation period, the colorforming ingredientwhich couples with the released anaphthol or other hydroxy substitutedaromatic compound is added. Any of the well-known azonium salts usefulin the manufacture of commercial dyes can be used, such as the azoniumsalts of aniline, o-dianisidine, 4-benzoylamino-2,5-dimethoxyaniline,4-amino-2,5-diethoxybenzanilide, nitrobenzene-azo 2,5 dimethoxyaniline,2-amino-4-chloroanisole, m-nitroaniline and the like. It has been foundthat tetrazotizcd o-dianisidine (Naphthanil Diazo Blue B) isparticularly effective since with this material an unusually deep coloris obtained with very small amounts of hydroxy aromatic compound.Normally about 0.2 to about 0.6 mg. of the azonium salt is added to theserum and substrate after completion of the incubation period. Thiscomponent is preferably added in the form of a solid tablet prepared bymixing the azonium salt with a filler such as sucrose, lactose and thelike and a lubricant followed by tableting in the conventional manner.For ease of handling, it is generally desirable that sutficient inertmaterial be incorporated to produce a tablet weighing at least 15 mg.

After the addition of the azonium salt, the color is allowed to developfor a fixed period of time and then compared with a calibrated colorstandard. The period allowed for color development is not critical, butit should preferably be the same as that used in the preparation of thestandards. Normally a time in the range of 2 to 4 minutes is preferred.

In the test procedure of the present invention, serum containing normalamounts of acid phosphatase will show a light yellow-brown color whenthe test is carried out using sodium a-naphthyl phosphate andtetrazotized odianisidine. Markedly elevated levels of serum acidphosphatase which indicate a suspicion of cancer of the prosstate show acolor of deep reddish purple. These differences in color are easilydetermined by even unskilled people with the result that the method ofthe invention affords a convenient and effective diagnostic tool.

In order further to illustrate this invention the following examples aregiven:

Example I 0.77 gram sodium a-naphthyl phosphate, 5.32 grams citric acidmonohydrate, 13.91 grams trisodium citrate dihydrate, 0.4 gram leucineand 0.6 gram Carbowax-6000 are well blended and formed into 21 milligramtablets, each containing 0.77 milligram sodium a-naphthyl phosphate and19.23 milligrams citrate buffer.

Example II 0.67 gram sodium wnaphthyl phosphate, 10 grams disodiumcitrate, 8.3 grams trisodium citrate dihydrate, 6 grams lactose and 2grams leucine are thoroughly blended and formed into 27 milligramtablets, each tablet containing 0.67 milligram sodium a-naphthylphosphate and 18.33 milligrams citrate buffer.

Example III 0.4 gram tetrazotized o-dianisidine, 4.2 grams of sucroseand 0.25 gram Carbowax-6000 are blended and formed into 5 milligramtablets, each tablet containing 0.4 milligram tetrazotizedo-dianisidine.

Example IV 0.4 gram tetrazotized o-dianisidine, 18.6 grams sucrose and 1gram Carbowax-6000 were blended and formed into 20 milligram tablets,each tablet containing 0.4 milligram tetrazotized o-dianisidine.

The tablets obtained by the procedures described in Examples I and IIare added before incubation to the serum whose acid phosphatase activityis to be determined. Tablets prepared in accordance with Examples IIIand IV are added to the incubated serum to develop the color, the depthof which is a measure of the acid phosphatase contained in the serumbeing tested.

Example V To approximately 0.2 millimeter of serum is added 1 tabletprepared in accordance with Example II. The tablet is broken up with aglass rod and the mixture is incubated at 72 F. for 20 minutes. To theincubated serum and substrate is then added a tablet prepared inaccordance with Example IV, the tablet being broken up with a glass rodand stirred into the serum. After exactly 3 minutes, the developed coloris matched against a standard color chart which has previously beenprepared from serum containing known amounts of acid phosphatase enzymeand an accurate reading of the acid phosphatase activity of the testserum is thus conveniently obtained.

Any-departure from the foregoing description which conforms to thepresent invention is intended to be included within the scope of theclaims.

I claim:

1. A substrate for use in the determination of prostatic acidphosphatase in serum which comprises a buffer adapted to maintain theserum pH within about 4.5 to about 5.5 and a salt of a-naphthylphosphate.

2. A substrate according to claim 1, which comprises about to about 25milligrams of said bufier and about 0.4 to about 1 milligram of saidsalt.

3. A substrate according to claim 2 wherein said bufier comprises saltsof acids selectd from the group consisting of citric acid, oxalic acidand malic acid and said salt is an alkali metal salt.

4. A substrate according to claim 3 wherein said salt is a sodium salt.

5. A dry preparation for use in the determination of prostatic acidphosphatase in serum which comprises about 0.4 to about 1 milligram of asalt of u-naphthyl phosphate and about 10 to about 25 milligrams of abuffer adapted to maintain the pH of the serum within about 4.5 to about5.5.

6. A dry preparation for use in the determination of serum acidphosphatase comprising about 10 to about 25 milligrams of a butleradapted to maintain the serum pH .vithin about 4.5 to about 5.5, saidbuffer comprising salts of acids selected from the group consisting ofcitric acid, oxalic acid and malic acid, and about 0.4 to about 1milligram of a metal salt of a phenolic phosphate ester, said metalbeing selected from the group consisting of the alkali and alkalineearth metals and said ester being the phosphoric acid ester of ahydroxy-substituted aromatic compound selected from the group consistingof benzene and naphthalene.

7. A method for the determination of prostatic acid phosphatase in serumwhich comprises incubating a small volume of serum in a substratecomprising a buffer adapted to maintain the pH of said serum withinabout 4.5 to about 5.5 and a salt of a-naphthyl phosphate, adding tosaid incubated mixture an azonium salt of an aromatic amine andcomparing the developed color to the color developed in serum in thepresence of a known concentration of prostatic acid phosphatase.

8. A method for the determination of prostatic acid phosphatase in serumwhich comprises adding to a small volume of serum a substrate comprisingabout 10 to about 25 milligrams of a buffer adapted to maintain the pHof said serum within about 4.5 to about 5.5, said bufier comprisingsalts of acids selected from the group consisting of citric acid, oxalicacid and malic acid, and about 0.4 to about 1 milligram of an alkalimetal salt of a-naphthyl phosphate, incubating the mixture of said serumand said substrate at a temperature of about 65 F. to 98 F. for about 25to about 12 minutes to permit the prostatic acid phopshatase enzymepresent to hydrolyze said a-naphthyl phosphate, adding to said incubatedmixture an azonium salt of an aromatic amine containing 1 to 3 NI-I,groups per molecule and comparing the developed color to the colordeveloped in serum in the presence of a known concentration'of prostaticacid phosphatase.

9. A method according to claim 8 wherein said bufier comprises salts ofcitric acid, said alkali metal salt of a-naphthyl phosphate is a sodiumsalt and said azonium salt is tetrazotized o-dianisidine.

10. A method for the determination of prostatic acid phosphatase inserum which comprises adding to a small volume of serum a first drypreparation comprising about 10 to about 25 milligrams of a citratebuffer adapted to maintain the pH of said serum within about 4.5 toabout 5.5 and about 0.4 to about 1 milligram of an alkali metal salt ofe-naphthyl phosphate, incubating the mixture of said serum and saidsubstrate at a temperature of about 65 F. to about 98 F. for about 25 toabout 12 minutes to permit the prostatic acid phosphatase enzyme presentto hydrolyze said ot-naphthyl phosphate, adding to said incubatedmixture a second dry preparation comprising about 0.2 to about 0.6milligram of tetrazotized o-dianisidine and comparing the developedcolor to the color developed in serum in the presence of a knownconcentration of prostatic acid phosphatase.

11. A method for the determination of serum acid phosphatase whichcomprises adding to a small volume of serum a first dry preparationcomprising a buffer adapted to maintain the pH of said scrum withinabout 4.5 to about 5.5 and a metal salt of a phenolic phosphate ester,said metal being selected from the group consisting of alkali andalkaline earth metals and said ester being a phosphoric acid ester of ahydroxy substituted aromatic compound selected from the group consistingof benzene and naphthalene, incubating the mixture to permit the acidphosphatase enzyme present to hydrolyze said phos phate ester, adding tosaid incubated mixture at second dry preparation comprising an azoniumsalt of an aromatic amine containing from 1 to 2 -NH, groups permolecule and comparing the developed color to the color developed inserum in the presence of a known concentration of acid phosphatase.

12. A method for the determination of serum acid phosphatase whichcomprises adding to a small volume of serum a first tablet comprisingabout 10 to about 25 milligrams of a bufier comprising salts of acidsselected from the group consisting of citric acid, oxalic acid and malicacid adapted to maintain the pH of said serum within about 4.5 to about5.5 and about 0.4 to about 1 milligram of an alkali metal salt of aphosphoric acid ester of a hydroxy substituted aromatic compoundselected from the group consisting of benzene and naphthalene,incubating the mixture of said serum and said first tablet at atemperature between 65 F. and 98 F. for about 25 to about 12 minutes topermit the acid phosphatase enzyme present to hydrolyze said ester,adding to said incubated mixture a second tablet comprising about 0.2 toabout 0.6 milligram of an azonium salt of an aromatic amine containingfrom 1 to 2 -NH; groups per molecule, and comparing the developed colorwith the color developed in serum in the presence of a knownconcentration of acid phosphatase.

13. A method according to claim 12 wherein said bufier is a citratebutter and said azonium salt is tetrazotized o-dianisidine.

Organic Chemistry," Fieser and Fieser, D. C. Heath & 00., 1944, Pp.859868.

1. A SUBSTRATE FOR USE IN THE DETERMINATION OF PHOSTATIC ACIDPHOSPHATASE IN SERUM WHICH COMPRISES A BUFFER ADAPTED TO MAINTAIN THESERUM PH WITHIN ABOUT 4.5 TO ABOUT 5.5 AND A SALT OF A-NAPHTHYLPHOSPHATE.
 7. A METHOD FOR THE DETERMINATION OF PORSTATIC ACIDPHOSPHATASE IN SERUM WHICH COMPRISES INCUBATING A SMALL VOLUME OF SERUMIN A SUBSTRATE COMPRISING A BUFFER ADAPTED TO MAINTAIN THE PH OF SAIDSERUM WITHIN ABOUT 4.5 TO ABOUT 5.5 AND A SALT OF A-NAPHTHYL PHOSPHATE,ADDING TO SAID INCUBATED MIXTURE AN AZONIUM SALT OF AN AROMATIC AMINEAND COMPARING THE DEVELOPED COLOR TO THE COLOR DEVELOPED IN SERUM IN THEPRESENCE OF A KNOWN CONCENTRATION OF PROSTATIC ACID PHOSPHATASE.